The Hsp90 family of molecular chaperones is required for the maturation, activation, and/or stability of diverse proteins that play central roles in malignant progression. As a result of this diversity, Hsp90 inhibitors simultaneously antagonize a wide variety of oncogenic pathways and processes. Thus, Hsp90 inhibitors are widely envisioned to have great potential as anti-cancer drugs. The Hsp90 inhibitors that have progressed to clinical trials share a common mechanism of action, namely binding to the ATP binding site in Hsp90's N- terminal domain and inhibiting Hsp90 function in vivo. Unfortunately, N-terminal Hsp90 inhibitors also have the untoward effect of inducing the pro-survival heat shock response, which is acknowledged to undermine the clinical efficacies of N-terminal Hsp90 inhibitors. Small molecules that bind to Hsp90's C-terminus have also been identified that inhibit Hsp90 function in vitro, and show cytostatic/cytotoxic activity in cultured cancer cell lines, but do not appear to induce the heat shock response. Thus, targeting the C-terminal domain of Hsp90 may be a superior strategy for anti-cancer therapies based on the inhibition of Hsp90. However, the progression of C-terminal Hsp90 inhibitors as clinical agents is hindered by two significant knowledge gaps: (i) The structural basis of their binding to Hsp90 is unknown, but must be determined for the rational design and optimization of novel inhibitory compounds; and (ii) the physiological basis of their anti-proliferative / cytotoxic activities is poorly understood. To further the development of C-terminal Hsp90 inhibitors with clinical potential, we propose to: 1) determine the structure of Hsp90 complexed to C-terminal Hsp90 inhibitors; and 2) compare and contrast the impacts of C-terminal vs. N-terminal inhibitors on the proteomes of breast cancer cells cultured as 3D spheroids. The structure of a N-terminally truncated Hsp90 construct bound to Hsp90 C- terminal inhibitors will be determined by X-ray crystallography. The impact of N- and C-terminal Hsp90 inhibitors on breast cancer cell proteomes will be characterized by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC), in conjunction with multi-dimensional liquid chromatography and a state-of-the-art Fusion Tribrid mass spectrometer. The proposed studies will provide critical structural information regarding the binding of C-terminal inhibitors to Hsp90, culminating in the rational design of C-terminal inhibitors with improved affinities for Hsp90 and clinical potential. The mechanistic insights into the selective tumoricidal activities of N- and C-terminal Hsp90 inhibitors generated by the proteomic studies will also reveal tumor- specific features that might guide chemotherapeutic decisions, tumor cell responses that might be monitored during clinical treatment, and promising modalities for combinatorial treatments with both families of Hsp90 inhibitors.